For genes involved in tissue generation and homeostasis, RNA polymerase II (Pol II) tends to pause immediately downstream of their transcription start sites. Compared to de novo recruitment of Pol II, a preassembled and poised Pol II could be more conducive to rapid and synchronous transcriptional activation in response to various physiologic cues. However, virtually nothing is known about the biological significance of this highly conserved and prevalent phenomenon in transcriptional regulation of tissue generation and homeostasis. We recently observed that many known adipogenic genes exhibit Pol II pausing. We also found that adipose tissue-specific ablation of a key Pol II-pausing factor, NELF, results in impaired white adipogenesis but elevated expression of genes involved in beige adipose thermogenesis. In the current proposal, we will test the central hypothesis that NELF-mediated Pol II pausing plays a dual function in white adipose tissue (WAT) homeostasis: it promotes differentiation of adipose progenitor cells to mature white adipocytes, and suppresses white-to-beige inter-conversion. Our multidisciplinary team of laboratory and clinician scientists will use mouse genetics, cell culture systems, and clinical samples to test this innovative hypothesis. We will first use murine and clinical samples to determine the cellular function of NELF in WAT establishment and maintenance. We will then use new mouse genetic models to evaluate the role of NELF in beige adipose thermogenesis. Lastly, we will combine target-gene approaches and genomic/bioinformatics tools to interrogate NELF-dependent Pol II pausing and transcriptional regulation in adipose homeostasis. The novel concept we seek to validate in our proposed work clearly departs from the prevailing transcription initiation-centric paradigm in adipose gene regulation. In particular, the new Pol II pausing-dependent mechanism points to a previously unappreciated direction for elucidating the transcriptional network that regulates adipose homeostasis. When successfully executed, our studies, underpinned by extensive preliminary data, promise to yield new insights into obesity-related metabolic diseases.